The present invention relates to a novel protein useful as a diagnostic tool for studies and researches relating to diagnostic and therapeutic applications to tumors, including uses in detecting tumor cells, estimating cancer malignancies, etc., and/or useful in other medical and physiological uses; and to a novel gene encoding said protein. More specifically, the present invention relates to a new membrane-type protein which is one of the MMPs having the activation capability of pro-matrix metalloproteinase 2 (pro-MMP-2), i.e. an activator for pro MMP-2, provided that said protein is different from the first membrane-type matrix metalloproteinase (MT-MMP-1), and to a gene coding for said protein. The present invention also encompasses a novel matrix metalloproteinase being specifically expressed in a human tumor cell surface layer (the instant novel matrix metalloproteinase is named xe2x80x9cmembrane-type matrix metalloproteinase-3 (MT-MMP-3)xe2x80x9d); DNA containing a nucleotide sequence coding the protein; a host cell transformed or transfected with the DNA, a process for producing the matrix metalloproteinase which comprises using said host cell, a monoclonal antibody capable of specifically binding with the matrix metalloproteinase protein, and applications of said protein and antibody.
An extracellular matrix may block the transfer of tumor cells in the invasion and metastasis of tumor cells that are present in a primary nest tissue. In order for tumor cells to transfer and invade into tissues, they must deviate from the primary nest and destroy peripheral extracellular matrixes. The metastasis of tumor cells progresses via destruction of basement membranes, invasion into and effusion from blood vessels, successful implantation on secondary organs, further growth, etc. The extracellular matrix that blocks tumor metastasis is composed of various complex components, including type IV collagen, proteoglycans, elastin, fibronectin, laminin, heparan sulfate, etc. A family of enzymes, generally named xe2x80x9cMatrix Metalloprotenasexe2x80x9d (hereinafter briefly referred to as xe2x80x9cMMPxe2x80x9d), with distinct substrate specificities are responsible for the degradation of the extracellular matrix.
It has been reported that MMP includes fibroblast-type collagenase (MMP-1), 72 kDa gelatinase (referred to as type IV collagenase or gelatinase A; MMP-2), 92 kDa gelatinase (referred to as type IV collagenase or gelatinase B; MMP-9), stromelysin-1 (MMP-3), matrilysin (MMP-7), neutrophilic collagenase (MMP-8), stromelysin-2 (MMP-10), stromelysin-3 (MMP-11), etc. (Crit. Rev. Oral. Biol. Med., 4: 197 to 250, 1993). These MMPs form a family, and the primary structure of genes has been already reported. The reported amino-acid sequences deduced from cDNA data of these MMPs are recognized to be homologous, which are constituted of an N-terminal signal peptide basically removed during secretion and processing, followed by a propeptide domain, a Zn+-binding catalytic domain, a proline-rich hinge domain composed of 5 to 50 amino acids, and a C-terminal hemopexin coagulation enzyme-like domain. There is no hemopexin-like domain in MMP-7. MMP-2 and MMP-9 include a gelatine-binding domain in addition to these.
Among these MMPs, it has been reported many times that type IV collagenase (MMP-2 and MMP-9) acting on, as a dominant substrate, type IV collagen that is a principal constituent for basement membranes is highly expressed in high metastatic tumor cells and there has been suggested that tumor cells are associated with tumor invasion into basement membrane invasion (Cell., 64: 327 to 336, 1991). The regulation of MMP activation is believed to be performed in steps including at least transcription level, a step for converting a proenzyme form wherein its enzymatic activity is latent into an active enzyme form, and controls by tissue inhibitor of metalloproteinase (TIMP) being a specific inhibitor against MMPs, etc. (Trends Genet., 6: 121 to 125, 1990).
All of the MMPs are secreted as inactive zymogens. In in vitro studies, activation of MMP-1 and MMP-9 is shown to be produced with serine proteinases such as plasmin, trypsin, cathepsin G. It has also been reported that activation of MMP-9 is caused by the action of active MMP-3 (J. Biol. Chem., 267: 3581 to 3584, 1992). However, since MMP-2 has no cleavage site sensitive to the above mentioned proteinase, activation of MMP-2 is believed not to be generated thereby (Curr. Opin. Cell Biol., 5: 891 to 897, 1993).
It has also been reported that these MMPs are produced by not only tumor cells but also circumferential fibroblasts and inflammatory cells which produce distinct MMPs, respectively (Breast Cancer Res. Treat., 24: 209 to 218, 1993; and Curr. Opin. Cell Biol., 5: 891 to 897, 1993).
It has previously been reported that, among them, MMP-2 is expressed in fibroblasts at a variety of sites accompanied with remodeling of tissue constructs and its activation is specifically generated in cancer tissues exemplified by lung cancer, in comparison with normal tissue and cancer tissue MMP-2s (Clin., Exp., Metastasis, 11: 183 to 189, 1993). In MMP-9, there is a low frequency that an active type is detected. In addition, there is proved in in vitro studies that active MMP-2 is localized at the apical site of tumor invasion (invadopodia) and it is suggested that the active MMP-2 has an important role on tumor invasion (Cancer Res., 53: 3159 to 3164, 1993; and Breast Cancer Res. Treat., 53: 3159 to 3164, 1994).
Under these backgrounds, attention has been focused on the activation mechanism of MMP-2. As described previously, however, activation of MMP-1 and MMP-9 is mediated by serine proteinases such as trypsin while the activation mechanism of MMP-2 is still undisclosed. In particular, an activating factor for MMP-2 remains unidentified. When HT1080 cells (MMP-2 producing cells) are treated with concanavalin A or 12-o-tetradecanoylphorbol 13-acetate (TPA), it is known that active MMP-2 appears in cultured medium, and it is believed that MMP-2 activating factors are induced in these cells (J. Natl. Cancer Inst., 85: 1758 to 1764, 1993; and Clin. Exp. Metastasis., 11: 183 to 189, 1993). Since this MMP-2 activation is induced by cellular membrane fractions and the activation is suppressed by chelating agents or TIMP, the MMP-2 activating factors have been presumed to be a membrane-type MMP (J. Biol. Chem., 268: 14033 to 14039, 1993).
The present inventors have previously cloned novel MMP genes using genetic engineering techniques, and obtained cloned genes coding for a new MMP having a typical transmembrane (TM) domain at the C-terminus thereof and being capable of activating MMP-2 (Nature, 370: 61 to 65, 1994). In fact, when this gene is expressed in cultured cells, the gene products are localized on the cell membrane without secretion. Thus, the present inventors have named such MMP as xe2x80x9cmembrane-type MMP (MT-MMP)xe2x80x9d.
Since, as described above, for MMPs, specifically MMP-2, the active form is found specifically in tumor cells, it is increasingly recognized that such should be targeted by anti-cancer or anti-metastatic drugs. Still, since MMP-2 exists relatively homeostatically as a zymogen in normal tissues, the regulation of MMP-2 activation resides in a process of activating it to active enzymes. Therefore, it is considered that the retrieval or identification of activating factors which are keys to this is extremely important in view of markers in the diagnosis of cancers and in the determination of malignancy and targets of anti-metastatic drugs against cancers.
In addition, it has been pointed out that MMP-2 may be involved in the cleavage of : xcex2-amyloid protein which is associated with the crisis of Alzheimer""s diseases. The xcex2amyloid protein is a part of amyloid protein precursors, xc2xc of xcex2-amyloid protein area is included in the membrane-spanning (or transmembrane) area of the amyloid protein precursor, and the rest is outside the cells. It has been recently disclosed that several metabolic pathways of amyloid protein precursors exist, one of which is a process including a cleavage of inner sites of the xcex2-amyloid protein area with a protease called xe2x80x9cxcex1-secretasexe2x80x9d and a discharge outside cells. It has been recently found that an amyloid protein-degrading activity is present in MMP-2, with the possibility that MMP-2 would function as xcex1-secretase or an extracellular xcex2-amyloid protein-degrading enzyme (Nature, 362 : 839, 1993). The xcex2-amyloid protein is the main component of senile macula observed in the brains of patients with Alzheimer""s diseases, and forms the core of senile macula by self-aggregation and deposition thereof. Since functional reduction of xcex2-amyloid protein-degrading enzymes may occur in the brain of the patient with Alzheimer""s diseases, attention is focused on MMP-2. Here, the key is a process for activating MMP-2. The MT-MMP previously identified by the present inventors (newly named xe2x80x9cMT-MMP-1xe2x80x9d herein) is believed to be an activating factor for MMP-2, but the existence of unknown MMPs such as MT-MMP-1 can be anticipated from the fact that a variety of components exists in the extracellular matrix. The existence of activating factors for MMP-2, other than MT-MMP-1, is still undeniable.
A primary object of the present invention is to provide novel proteins which (i) belong to a member of MMPs having the capability of activating pro MMP-2, (ii) are different from MT-MMP-1, (iii) have the capability of activating pro MMP-2, and (iv) are an activator for pro MMP-2; genes encoding the same; processes for producing said novel pro MMP-2 activating factor proteins; applications of the protein and gene, etc.
The present inventors have observed that an activating factor (activator) for pro MMP-2 is assumed as a member of membrane-type MMPs since activation of pro MMP-2 is induced by tumor cell membrane fractions and the activation is inhibited by chelating agents or TIMP; the present inventors thus have isolated the gene coding for novel MMP-2 capable of activating pro MMP-2 in the prior research. However, the present inventors have hypothesized the existence of MMP acting as a MMP-2 activator in addition to the above, or MMP biochemically differing from the known MMPs. Following various researches using genetic engineering techniques, the present inventors successfully isolated a gene coding for MMP that is a novel activating factor for pro MMP-2, and completed the present invention.
It has been known that MT-MMP-1 is a member of MMPs capable of activating pro MMP-2; however, other activating factors for pro MMP-2 have been neither isolated nor identified. The present inventors have now cloned novel MMP genes, i.e. pro MMP-2 activating factor genes, and disclosed an entire nucleotide sequence of the gene and an entire amino acid sequence thereof. The inventors originally named this novel MMP as xe2x80x9cMT-MMP-2xe2x80x9d (Japanese Patent Application, Nos. Hei 7-200319 (or JP Appln. No. 200319/1995) and 7-200320 (or JP Appln. No. 200320/1995), both filed on Jul. 14, 1995). Later, at the Gordon Research Conference on Matrix Metalloproteinases (Andover, N.H., Jul. 16-21, 1995), it was agreed upon renaming xe2x80x9cMT-MMP-3xe2x80x9d (The Journal of Biological Chemistry, Vol. 270, pp. 23013-23020 (1995)). Therefore, the instant xe2x80x9cMT-MMP-3xe2x80x9d indicates the substance identical with MT-MMP-2 as described in Japanese Patent Application Nos. 7-200319 and 7-200320.
The present invention relates to novel proteins, i.e. MT-MMP-3 and analogs thereof. Further, the present invention relates to novel DNA sequences coding for all or part of MT-MMP-3, to vectors having such DNA sequences, and to host cells transformed or transfected with such vectors. The present invention also includes the production of recombinant MT-MMP-3 and uses of said recombinant MT-MMP-3. The present invention relates to antibodies which specifically bind with MT-MMP-3. In another aspect, the present invention relates to reagents for measurement or assay which contain said product and to detecting, measuring or assaying methods using such reagents. In particular, methods for detecting or measuring MT-MMP-3 in vivo and in vitro are provided.
The present invention relates to (1) proteins or a salt thereof which (i) belong to a member of MMPs capable of activating pro MMP-2 but are not MT-MMP-1, (ii) are an activator for pro MMP-2 and (iii) have an activity identical with or substantially equivalent to naturally-occurring MT-MMP, or a salt thereof; (2) characteristic partial peptides of said protein or a salt thereof; (3) genes (for example, nucleic acids including DNA, RNA, etc.) coding for said protein or peptide; (4) vectors or plasmids which contain said gene operably with gene recombination techniques; (5) host cells transformed or transfected with such vectors or the like; (6) processes for producing said protein or a salt thereof which comprises culturing said transformed or transfected host cell (transformant or transfectant); (7) antibodies (in particular, monoclonal antibodies) obtained using a member selected from the group consisting of the protein or a salt thereof thus obtained in the above process and the characteristic partial peptide of the protein or a salt thereof thus obtained in the above process; (8) hybridoma cells which produce the antibody; and (9) measuring (assaying) and/or diagnostic means (i) using the isolated gene (including, for example, DNA, RNA, etc.) as a probe or (ii) using the antibody.
Particularly, the present invention relates to (1) proteins which (i) belong to a member of MMPs capable of activating pro MMP-2, (ii) are an activator for pro MMP-2 but different from MT-MMP-1 and (iii) have an activity identical with or substantially equivalent to native MT-MMP-3, or a salt thereof; (2) characteristic partial peptides of said protein or a salt thereof; (3) genes (including, for example, DNA, RNA, etc.) coding for said protein or peptide; (4) vectors or plasmids wherein said gene is contained operably with gene recombination techniques; (5) host cells transformed or transfected with such a vector or the like; (6) processes for producing said protein or a salt thereof which comprises culturing said transformed or transfected host (transformant or transfectant)); (7) antibodies (in particular, monoclonal antibodies) obtained using a member selected from the group consisting of said protein or a salt thereof thus obtained and the unique partial peptide of said protein or a salt thereof thus obtained; (8) hybridoma cells which produce the antibody; and (9) measurement (assay) and/or diagnosis means (i) using the isolated gene (including, for example, DNA, RNA, etc.) as a probe or (ii) using the antibody.
Preferably, the present invention is related to MT-MMP-3 or a salt thereof which has (i) an amino acid sequence represented by SEQ ID NO: 2 in the Sequence Listing or (ii) an amino acid sequence substantially equivalent to SEQ ID NO: 2.
The present invention provides:
(1) a protein or a salt thereof, which (i) belongs to a member of MMPs having the activation capability of pro MMP-2, (ii) has an activity identical with or substantially equivalent to naturally-occurring MT-MMP, and (iii) is a pro MMP-2 activating factor, excluding MT-MMP-1;
(2) the protein according to above (1), wherein the protein has a biological property or primary structural conformation identical with or substantially equivalent to that of native MT-MMP-3 or a salt thereof;
(3) the protein according to above (1) or (2), wherein a C-terminal area of the protein has (i) an amino acid sequence from Ala564 to Phe587 in the sequence represented by SEQ ID NO: 2 in the Sequence Listing or (ii) an amino acid sequence substantially equivalent thereto;
(4) the protein according to any of above (1) to (3), wherein the protein is MT-MMP-3 or a salt thereof which has (i) an amino acid sequence represented by SEQ ID NO: 2 in the Sequence Listing or (ii) an amino acid sequence equivalent thereto;
(5) the protein according to any of above (1) to (4), wherein the protein is the product of prokaryotic or eukaryotic expression of an exogenous DNA sequence;
(6) the protein according to any of above (1) to (5), wherein the protein has (i) the amino acid sequence of SEQ ID NO: 2 in the Sequence Listing or (ii) the substantially same amino acid sequence;
(7) a partial peptide (or a peptide fragment) of the protein according to any of above (1) to (6) or a salt thereof;
(8) a nucleic acid comprising a nucleotide sequence coding for the protein or the partial peptide according to any of above (1) to (7);
(9) the nucleic acid according to above (8) which is a DNA gene having a nucleotide sequence coding for MT-MMP-3 according to any of above (2) to (4);
(10) the nucleic acid according to above (8) or (9), having (i) an open reading frame region of the nucleotide sequence represented by SEQ ID NO: 1 in the Sequence Listing or (ii) a nucleotide sequence having an activity substantially equivalent thereto;
(11) a vector comprising the nucleic acid according to any of above (8) to (10);
(12) a transformant or transfectant harboring (i) the nucleic acid according to any of above (8) to (10) or (ii) the vector according to above (11);
(13) a process for producing the protein according to any of above (1) to (6) or a partial peptide thereof, which comprises:
(i) culturing the transformant or transfectant according to above (12) in a nutrient medium capable of growing said transformant or transfectant, and
(ii) producing, as a recombinant species, the protein according to any of above (1) to (6) or a partial peptide thereof, including MT-MMP-3 or a salt thereof;
(14) an antibody against (a) a protein or a salt thereof which (i) belongs to a member of MMPs having the activation capability of pro MMP-2, (ii) has an activity identical with or substantially equivalent to naturally-occurring MT-MMP, and (iii) is a pro MMP-2 activating factor, excluding MT-MMP-1, or (b) a partial peptide of said protein or a salt thereof;
(15) the antibody according to above (14), wherein the antibody is against the protein which has an activity or a primary structural conformation identical with or substantially equivalent to that of MT-MMP-3 or a salt thereof;
(16) the antibody according to above (14) or (15), wherein the antibody is against the protein that is MT-MMP-3 or a salt thereof having (i) an amino acid sequence represented by SEQ ID NO: 2 in the Sequence Listing or (ii) an amino acid sequence substantially equivalent thereto;
(17) the antibody according to any of above (14) to (16), wherein the antibody is against the protein which is a product obtained by expressing a foreign DNA sequence in prokaryotic or eukaryotic cells;
(18) the antibody according to any of above (14) to (17), wherein the antibody is against the protein which has (i) the amino acid sequence of SEQ ID NO: 2 in the Sequence Listing or (ii) the substantially same amino acid sequence;
(19) the antibody according to any of above (14) to (18), wherein the antibody is against a partial peptide of the protein or a salt thereof;
(20) the antibody according to any of above (14) to (19), wherein the antibody is an anti-serum;
(21) the antibody according to any of above (14) to (19), wherein the antibody is monoclonal;
(22) the antibody according to any of above (14) to (19) and (21), which is a monoclonal antibody against MT-MMP-3 or a salt thereof;
(23) a method for producing an antibody against (a) a protein or a salt thereof which (i) belongs to a member of MMPs having the activation capability of pro MMP-2, (ii) has an activity identical with or substantially equivalent to naturally-occurring MT-MMP, and (iii) is a pro MMP-2 activating factor, excluding MT-MMP-1, or (b) a partial peptide of said protein or a salt thereof, which comprises employing an antigen selected from the group consisting of said protein, said partial peptide and a salt thereof to raise the antibody thereagainst;
(24) a method for producing the antibody according to above (21) or (22), which comprises
(A) fusing an antibody-producing cell obtained from an immunized animal with an immortal cell, wherein said antibody is against (a) a protein or a salt thereof which (i) belongs to a member of MMPs having the activation capability of pro MMP-2, (ii) has an activity identical with or substantially equivalent to naturally-occurring MT-MMP, and (iii) is a pro MMP-2 activating factor, excluding MT-MMP-1, or (b) a partial peptide of said protein or a salt thereof and said animal is immunized with the protein, the partial peptide or a salt thereof, and
(B) selecting an immortal hybrid cell capable of an antibody against a protein including MT-MMP-3;
(25) a method for detecting and/or measuring MT-MMP-3, which comprises using (A) a reagent selected from the group consisting of (a) a protein or a salt thereof which (i) belongs to a member of MMPs having the activation capability of pro MMP-2, (ii) has an activity identical with or substantially equivalent to naturally-occurring MT-MMP, and (iii) is a pro MMP-2 activating factor, excluding MT-MMP-1, and (b) a partial peptide of said protein or a salt thereof, or (B) a reagent selected from the group consisting of the antibodies according to any of above (14) to (22);
(26) a labeled antibody against MT-MMP-3 for the method for detecting and/or measuring MT-MMP-3 (the detection and/or measurement of MT-MMP-3) according to above (25);
(27) a labeled protein or a salt thereof, for the method for detecting and/or measuring MT-MMP-3 (the detection and/or measurement of MT-MMP-3) according to above (25), wherein said labeled protein (i) belongs to a member of MMPs having the activation capability of pro MMP-2, (ii) has an activity identical with or substantially equivalent to naturally-occurring MT-MMP, and (iii) is a pro MMP-2 activating factor, excluding MT-MMP-1, or a labeled partial peptide of said protein or a salt thereof, for the method according Lo above (25);
(28) a labeled nucleic acid for detection and/or measurement of MT-MMP-3 expressing cells and/or tissues, wherein said nucleic acid encodes (A) a protein which (i) belongs to a member of MMPs having the activation capability of pro MMP-2, (ii) has an activity identical with or substantially equivalent to naturally-occurring MT-MMP, and (iii) is a pro MMP-2 activating factor, excluding MT-MMP-1, or (B) a partial peptide of said protein; and
(29) the nucleic acid according to above (28), which is a probe for hybridization.
In particular, the present invention provides:
(30) MT-MMP-3 or a salt thereof which has an amino acid sequence represented by SEQ ID NO: 2 in the Sequence Listing or an amino acid sequence substantially equivalent thereto;
(31) a partial peptide of MT-MMP-3 or a salt thereof according to above (30);
(32) a DNA gene comprising a nucleotide sequence coding for MT-MMP-3 according to above (30);
(33) the DNA gene according to above (32), which has a nucleotide sequence represented by SEQ ID NO: 1 in the Sequence Listing;
(34) a vector comprising the gene according to above (32);
(35) a transformant (or transformed cell) harboring (i) the gene according to above (32) or (ii) the vector according to above (34);
(36) a process for producing MT-MMP-3 or a salt thereof, which comprises culturing the transformant according to above (35) in a nutrient medium capable of growing said transformant to produce, as a recombinant protein, said MT-MMP-3 or a salt thereof;
(37) a process for producing an antibody against MT-MMP-3 or a salt thereof, which comprises using an antigen selected from the group consisting of MT-MMP-3 or a salt thereof according to above (30) and a partial peptide of said MT-MMP-3 or a salt thereof to raise the antibody thereagainst;
(38) an antibody against MT-MMP-3 according to above (31);
(39) the antibody (anti-MT-MMP-3 antibody) according to above (38), which is anti-serum;
(40) the antibody (anti-MT-MMP-3 antibody) according to above (38), which is monoclonal;
(41) a process for producing a monoclonal antibody against MT-MMP-3 (monoclonal anti-MT-MMP-3 antibody; anti-MT-MMP-3 mAb) according to above (40), which comprises fusing an anti-MT-MMP-3 antibody-producing cell with an immortal cell and selecting an immortal hybrid cell (hybridoma cell) capable of producing anti-MT-MMP-3 mAb, wherein said anti-MT-MMP-3 antibody-producing cell is obtained from an animal immunized with a member selected from the group consisting of MT-MMP-3 or a salt thereof according to above (30) and a partial peptide of said MT-MMP-3 or a salt thereof;
(42) a method for detecting and/or measuring MT-MMP-3, which comprises using (A) a reagent selected from the group consisting of MT-MMP-3 or a salt thereof according to above (30) and a partial peptide of said MT-MMP-3 or a salt thereof, or (B) a reagent selected from the group consisting of anti-MT-MMP-3 antibodies according to above (38);
(43) Labeled MT-MMP-3 or a salt thereof, or a labeled partial peptide of MT-MMP-3, for the method for detecting and/or measuring MT-MMP-3 according to above (42); and
(44) a labeled antibody against MT-MMP-3 (labeled anti-MT-MMP-3 antibody) for the method for detecting and/or measuring MT-MMP-3 according to above (42).